This invention relates to compounds which inhibit the interaction between MDM2 and the tumour suppressor protein, p53. This invention also relates to processes for the manufacture of MDM2/p53 interaction inhibitors and pharmaceutically acceptable salts, prodrugs or solvates thereof, to novel pharmaceutical compositions containing them and to the use of the compounds as probes of MDM2 and p53 function.
p53 is a transcription factor which plays a pivotal role in the regulation of the balance between cell proliferation and cell growth arrest/apoptosis. Under normal conditions the half life of p53 is very short and consequently the level of p53 in cells is low. However, in response to cellular DNA damage or cellular stress, levels of p53 increase. This increase in p53 levels leads to the activation of the transcription of a number of genes which induces the cell to either growth arrest or to undergo the processes of apoptosis. Thus the function of p53 is to prevent the proliferation of transformed cells and thus protect the organism from the development of cancer (for a review see Levine 1997, Cell 88, 323-331).
MDM2 is a key negative regulator of p53 function, which binds to the amino terminal transactivation domain of p53. MDM2 both inhibits the ability of p53 to activate transcription and targets p53 for proteolytic degradation, thus maintaining the low levels of p53 under normal conditions. MDM2 may also have separate functions in addition to inhibition of p53. For example, MDM2 also binds another tumour suppressor protein, the retinoblastoma gene product, and inhibits its ability to activate transcription. For reviews of MDM2 function see: Piette et al (1997) Oncogene 15, 1001-1010; Lane and Hall (1997) TIBS 22, 372-374; Lozano and de Oca Luna (1998) Biochim Biophys Acta 1377, M55-M59.
MDM2 is a cellular proto-oncogene. Overexpression of MDM2 has been observed in a range of cancers see Momand and Zambetti (1997) J. Cell. Biochem 64, 343-352. The mechanism by which MDM2 amplification promotes tumourigenesis is at least in part related to its interaction with p53. In cells over-expressing MDM2 the protective function of p53 is blocked and thus cells are unable to respond to DNA damage or cellular stress by increasing p53 levels, leading to cell growth arrest and/or apoptosis. Thus after DNA damage and/or cellular stress cells over-expressing MDM2 are free to continue to proliferate and assume a tumourigenic phenotype. Under these conditions disruption of the interaction of p53 and MDM2 would release the p53 and thus allow the normal signals of growth arrest and/or apoptosis to function. Thus disruption of the interaction of MDM2 and p53 offers an approach for therapeutic intervention in cancer.
A few patent applications have been published which describe peptide inhibitors of the interaction of p53 and MDM2, see: International Patent Application, WO9602642, University of Dundee; International Patent Application WO 9801467, Novartis and Cancer Research Campaign Technology Ltd.; and International Patent Application WO 9709343.
We have discovered a novel class of small molecule compounds which inhibit the interaction of MDM2 and p53. These compounds are useful as probes of MDM2 and p53 function and may be useful as agents for the treatment of cancer.
According to the invention there is provided a compound of formula (1): 
wherein:
L1 is hydrogen or methyl;
R1 and R2 and R3 are each independently hydrogen, halo, nitro, cyano, carbamoyl, N-(C1-4alkyl)carbamoyl, NN-(diC1-4alkyl)carbamoyl or C1-4alkoxycarbonyl;
R4 is indole, N-(C1-4 alkyl) indole, C5-7carbocyclic ring or aryl, any of which can be optionally substituted on ring carbon atoms with up to three substituents each independently selected from halo, C1-4alkyl, or C1-4alkoxy;
R5 is hydrogen, C1-4alkyl, R6CH2xe2x80x94 or R6C(O)xe2x80x94;
R6 is aryl, heteroaryl, heterocyclyl, aminoC3-6alkyl, N(C1-4alkyl)aminoC3-6alkyl, NN-(diC1-4alkyl)aminoC3-6alkyl, or R7; wherein the aryl, heteroaryl or heterocyclyl rings may be optionally substituted with up to three substituents independently selected from nitro, C1-4alkyl, C1-4alkoxy, halo, (C1-4alkyl)sulfanyl, C1-4 alkoxycarbonyl, N-(C1-4 alkyl)carbamoyl, NN-(diC1-4 alkyl)carbamoyl, N-(C1-4 alkyl)amino or NN-(diC1-4 alkyl)amino;
wherein R7 is either a group of formula (2) of formula (3): 
wherein:
L2, L3 and L4 are each independently hydrogen or methyl;
R8 is amino, guanadino, imidazolo, any of which can be mono or di-N-substituted with C1-4alkyl;
A1 is oxygen or a direct bond;
R9 is a C5-8 membered mono-carbocyclic ring, a C6-10 membered bi-carbocyclic ring, C8-12 membered tri-carbocyclic ring, C5-7alkyl or aryl, any of which can be optionally mono, bi or tri substituted by C1-4 alkyl,
R10 is C1-6alkyl or a C3-8mono-carbocyclic ring;
R11 is hydrogen, halo, C1-4alkyl, or C1-4alkoxy;
R12 is hydrogen or methyl or ethyl or R12 together with L2 forms a C5-7 nitrogen-containing heterocyclic ring;
R13 is hydrogen or methyl ethyl or R13 together with L4 forms a C5-7 nitrogen-containing heterocyclic ring;
n is 0, 1 or 2;
p is 0, 1 or 2;
q is an integer from 1 to 6
r is 0, 1 or 2;
s is 0, 1 or 2;
provided that when R6 is aryl, heteroaryl, heterocyclyl aminoC3-6alkyl, N-(C1-4alkyl)aminoC3-6alkyl or NN-(diC1-4alkyl)aminoC3-6alkyl then R6 is other than R6CH2xe2x80x94; and when R1 is hydrogen, R2 is hydrogen, R3 is hydrogen, L1 is hydrogen, n is 1, R4 is phenyl, R5 is R6C(O)xe2x80x94, then R6 cannot be 2-methyl-4-amino-butyl,
or a pharmaceutically acceptable salt, prodrug or solvate thereof.
In this specification the generic term xe2x80x9calkylxe2x80x9d includes both straight-chain and branched-chain alkyl groups. However references to individual alkyl groups such as xe2x80x9cpropylxe2x80x9d are specific for the straight-chain version only and references to individual branched-chain alkyl groups such as xe2x80x9cisopropylxe2x80x9d are specific for the branched-chain version only. An analogous convention applies to other generic terms.
The term xe2x80x9carylxe2x80x9d refers to phenyl or naphthyl.
The term xe2x80x9cheteroarylxe2x80x9d refers to a 5-10 membered aromatic mono or bicyclic ring containing up to 5 heteroatoms selected from nitrogen, oxygen or sulphur. Examples of 5- or 6-membered heteroaryl ring systems include imidazole, triazole, pyrazine, pyrimidine, pyridazine, pyridine, isoxazole, oxazole, isothiazole, thiazole, furan, pyrazole, 1,2,3-thiadiazole and thiophene. A 9 or 10 membered bicyclic heteroaryl ring system is an aromatic bicyclic ring system comprising a 6-membered ring fused to either a 5 membered ring or another 6 membered ring. Examples of 5/6 and 6/6 bicyclic ring systems include benzofuran, benzimidazole, benzthiophene, benzthiazole, benzisothiazole, benzoxazole, benzisoxazole, pyridoimidazole, pyrimidoimidazole, quinoline, isoquinoline, quinoxaline, quinazoline, phthalazine, cinnoline, 4-oxo-4H-1-benzopyran and naphthyridine.
The term xe2x80x9cheterocyclylxe2x80x9d refers to a 5-10 membered non-aromatic mono or bicyclic ring containing up to 5 heteroatoms selected from nitrogen, oxygen or sulphur. Examples of xe2x80x98heterocyclylxe2x80x99 include pyrrolinyl, pyrrolidinyl, morpholinyl, piperidinyl, piperazinyl, dihydropyridinyl and dihydropyrimidinyl
The term xe2x80x9ccarbocyclic ringxe2x80x9d refers to a totally saturated or partially saturated mono, bi or tri cyclic carbon ring. Examples of carbocyclic rings are cyclopentyl, cyclohexyl, cyclopentyl, bicyclo-octane or adamantyl.
The term xe2x80x9chaloxe2x80x9d refers to fluorine, chlorine, bromine or iodine.
The term carbamoyl refers to xe2x80x94C(O)NH2.
The term xe2x80x9cwarm-blooded animalxe2x80x9d includes human.
Examples of C1-4alkyl include methyl, ethyl, propyl, isopropyl, sec-butyl and tert-butyl; examples of C1-4alkoxy include methoxy, ethoxy and propoxy; examples of C1-4alkanoyl include formyl, acetyl and propionyl; examples of C1-4alkylamino include methylamino, ethylamino, propylamino, isopropylamino, sec-butylamino and tert-butylamino; examples of di-(C1-4alkyl)amino include di-methylamino, di-ethylamino and N-ethyl-N-methylamino; examples of N-(C1-4alkyl)aminoC1-4alkyl include N-methyl-aminomethyl and N-ethyl-aminoethyl, and examples of NN-di(C1-4alkyl)aminoC1-4alkyl include: NN-dimethyl-aminomethyl and N-methyl-N-ethyl aminomethyl.
Examples of suitable values for R1, R2 or R3 include hydrogen, halo, nitro, carbamoyl, N-(C1-4alkyl)carbamoyl, NN-(diC1-4alkyl)carbamoyl or C1-4alkoxycarbonyl. Preferably hydrogen, halo, nitro, carbamoyl, N-ethylcarbamoyl, NN-dimethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methoxycarbonyl or ethoxycarbonyl. More preferably hydrogen, halo or nitro. More preferably hydrogen, chloro or nitro. Most preferably either R1 is halo, preferably chloro, R2 is halo, preferably chloro and R3 is hydrogen or R1 is hydrogen, R2 is nitro and R3 is hydrogen.
Examples of suitable values for R4 include indole, N-(C1-4alkyl) indole, C5-7carbocyclic ring or aryl, either of which can be optionally substituted by halo, C1-4alkyl, or C1-4alkoxy. Preferably R4 is indole, N-methylindole, cyclopentyl, cyclohexyl, phenyl or naphthyl, optionally substituted as defined above. More preferably R4 is indole, N-methylindole, cyclohexyl, or phenyl, optionally substituted as defined above. More preferably R4 is indole, N-methylindole, or phenyl, optionally substituted as defined above. More preferably R4 is phenyl, optionally substituted as defined above. Most preferable R4 is phenyl substituted with halo, preferably chloro.
Examples of suitable values for R5 include hydrogen, C1-4alkyl, R6CH2xe2x80x94 or R6C(O)xe2x80x94. Preferably R5 is hydrogen, methyl, R6CH2xe2x80x94 or R6C(O)xe2x80x94. Most preferably R5 is R6C(O)xe2x80x94.
Examples of suitable values for R6 include aryl, heteroaryl, heterocyclyl, amino-C3-6alkyl, N-(C1-4alkyl)aminoC3-6alkyl, NN-(diC1-4alkyl)aminoC3-6alkyl, or R7. Preferably R6 is aryl, heteroaryl, heterocyclyl, (NN-dimethyl)aminopropyl or R7. More preferably R6 is aryl, heteroaryl, heterocyclyl or R7. More preferably R6 is aryl and heteroaryl. Most preferably R6 is aryl, preferably phenyl.
Examples of substituents on the aryl ring systems of R6 include nitro, C1-4alkyl, C1-4alkoxy, halo, (C1-4alkyl)sulfanyl. Preferably nitro, C1-4alkyl, C1-4alkoxy or methylsulfanyl. More preferably nitro or C1-4alkoxy. Most preferably nitro or methoxy. More preferable substitution patterns on a phenyl group in R6 are 2-nitro-4,5-dimethoxy, 2-methyl-3-nitro, 2-methoxy, 2,4-dimethyl, 4,5-dimethoxy-2-nitro or 2-methylsulfanyl. Most preferred is 2-nitro-4,5-dimethoxy.
Examples of suitable values for heteroaryl rings at R6 include: furan, thiophene, pyrrole, pyrazole, isoxazole, thiazole, thiadiazole, pyridine, pyridazine, benzofuran, 4-oxo 4H-1-benzopyran. Heteroaryl ring systems with one or two heteroatoms are preferred. Heteroaryl ring systems with one heteroatom are most preferred.
Examples of suitable values for substituents on ring carbon atoms in heterocyclic rings of R6 include, C1-4alkyl, C1-4alkoxy, halo, hydroxy, oxo or C1-4alkylcarbonyl.
Examples of suitable values for R8 include amino, guanadino and imidazolo, any of which can be mono or di-N-substituted with C1-4alkyl. More preferably R8 is guanadino or amino, substituted as defined above. Preferably R8 is N-(C1-4alkyl)amino or amino. Most preferably R8 is amino.
Examples of suitable values for R9 include C5-7alkyl, a C5-10 membered carbocyclic ring and aryl, any of which can be optionally substituted with C1-4alkyl. Preferably R9 is 3-propylbutyl, 2,2 dimethylpropyl, a C5-10 membered carbocyclic ring, or aryl. More preferably R9 is cyclohexyl, cycloheptyl, adamantyl or aryl. Even more preferably R9 is cyclohexyl or aryl. Yet more preferably R9 is aryl. Most preferably R9 is phenyl.
Examples of suitable values for R10 include C1-4alkyl or a C3-7carbocyclic ring. More preferably R10 is a C3-7carbocyclic ring or a branched C1-4alkyl chain. Even more preferably R10 is a C3-7carbocyclic ring. Most preferably R10 is cyclohexyl.
Examples of suitable values for R11 include hydrogen, halo, C1-4alkyl, or C1-4alkoxy. More preferably R11 is hydrogen or C1-4alkyl. Most preferably R11 is hydrogen.
Examples of suitable values for R12 are hydrogen or methyl or when R12 is xe2x80x94CH2xe2x80x94, L2 is hydrogen and s is 1, R12 together with L2 forms a six membered nitrogen containing ring Preferably R12 and L2 are both hydrogen.
Examples of suitable values for R13 are hydrogen or methyl or when R13 is xe2x80x94CH2xe2x80x94, L4 is hydrogen and s is 1, R13 together with L4 forms a six membered nitrogen containing ring Preferably R13 and L4 are both hydrogen.
Preferably n is 1
Preferably p is 1
Preferably q is 2-4. Most preferable q is 4.
Preferably r is 1.
Preferably s is 1.
It is to be understood that, insofar as certain of the compounds of Formula (1) defined above may exist in optically active or racemic forms by virtue of one or more asymmetric carbon atoms, the invention includes in its definition any such optically active or racemic form which possesses the property of inhibiting MDM2 interactions. The synthesis of optically active forms may be carried out by standard techniques of organic chemistry well known in the art, for example by synthesis from optically active starting materials or by resolution of a racemic form. Similarly, inhibitory properties against MDM2 interactions may be evaluated using the standard laboratory techniques referred to hereinafter.
When R6 is other than R7 then the following stereo-chemistry is preferred, in the group xe2x80x94N(L1)CH((CH2)nR4)COxe2x80x94, in formula (1): 
A particular group of compounds of the invention include for example, a compound of formula (1) wherein R5 is hydrogen or C1-4 alkyl, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein either:
(a) R1 is hydrogen; R2 is nitro; R3 is hydrogen; R4 is indole, N-(C1-4alkyl)indole or cyclohexyl; R5 is hydrogen or methyl; L1 is hydrogen and n is 1 or 2; and where the optical centre represented in the Formula (X) is in the S configuration; or
(b) R1 is halo; R2 is halo; R3 is hydrogen; R4 is indole, N-(C1-4alkyl)indole or cyclohexyl; R5 is hydrogen or methyl; L1 is hydrogen and n is 1 or 2; and where the optical centre represented in the Formula (X) is in the S configuration.
A further preferred group of compounds of the invention include, for example a compound of formula (1) wherein R5 is R6C(O)xe2x80x94 or R6CH2xe2x80x94, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein either:
(a) R1 is hydrogen; R2 is nitro; R3 is hydrogen; R4 is indole; N-(C1-4alkyl)indole, halo-phenyl or cyclohexyl; R5 is R6C(O)xe2x80x94 or R6CH2xe2x80x94; R6 is substituted phenyl; L1 is hydrogen or methyl and n is 1 or 2; and where the optical centre represented in the Formula (X) is in the S configuration; or
(b) R1 is hydrogen; R2 is nitro; R3 is hydrogen; R4 is indole; N-(C1-4alkyl)indole; halo-phenyl or cyclohexyl, R5 is R6C(O)xe2x80x94 or R6CH2xe2x80x94, R6 is a substituted heterocycle; L1 is hydrogen or methyl and n is 1 or 2; and wherein the optical centre represented in the Formula (X) is in the S configuration.
A further preferred group of compounds of the invention include, for example a compound of formula (1) wherein R5 is R6C(O)xe2x80x94 and R6 is R7, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein either:
(a) R1 is hydrogen; R2 is nitro; R3 is hydrogen; R4 is indole; N-(C1-4alkyl)indole; cyclohexyl or halo-phenyl; R5 is R6C(O)xe2x80x94; R6 is R7; R7 is of formula (2); R8 is amino; R9 is an optionally substituted aryl group; R11 is hydrogen; A1 is oxygen or a bond; n is 1 or 2; p is 1; q is 2-4 and L1 and L2 and L3 are independently hydrogen or methyl; or
(b) R1 is hydrogen; R2 is nitro; R3 is hydrogen; R4 is indole; N-(C1-4alkyl)indole; cyclohexyl or halo-phenyl; R5 is R6C(O)xe2x80x94; R6 is R7; R7 is of formula (2); R8 is amino; R9 is an optionally substituted heterocyclic group; R11 is hydrogen; A1 is oxygen or a bond; n is 1 or 2; p is 1; q is 2-4 and L1 and L2 and L3 are independently hydrogen or methyl.
A further preferred group of compounds of the invention include, for example a compound of formula (1) wherein R5 is R6CH2xe2x80x94 and R6 is R7, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein:
(a) R1 is hydrogen; R2 is nitro; R3 is hydrogen; R4 is indole; N-(C1-4alkyl)indole; cyclohexyl or halo-phenyl; R5 is R6CH2xe2x80x94; R6 is R7; R7 is of formula (3); R10 is either a C3-7 carbocyclic ring or a C1-4alkyl chain; R11 is hydrogen; L4 is hydrogen or methyl and r is 1.
A more preferred compound of the invention is a compound of formula (1) wherein R5 is R6C(O)xe2x80x94 and R6 is a substituted phenyl group, or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein;
R1 is hydrogen; R2 is nitro; R3 is hydrogen; R4 is indole; N-(C1-4alkyl)indole; or 4-chloro-phenyl; R5 is R6C(O)xe2x80x94; R6 is 4,5-dimethoxy-2-nitrophenyl; L1 is hydrogen or methyl; n is 1 and where the optical centre represented in the Formula (X) is in the S configuration.
A further more preferred compound of the invention is a compound of formula (1) wherein R5 is R6C(O)xe2x80x94 and R6 is R7 and R7 is of formula (2), or a pharmaceutically acceptable salts, prodrugs or solvates thereof, wherein:
R1 is hydrogen; R2 is nitro; R3 is hydrogen; R4 is indole; N-(C1-4alkyl)indole; phenyl; 4-chloro-phenyl or cyclohexyl; R5 is R6C(O)xe2x80x94; R6 is R7; R7 is of formula (2); R8 is amino; A1 is oxygen or a bond, R9 is phenyl or cyclohexyl; R11 is hydrogen; L1, L2 and L3 are independently hydrogen or methyl; n is 1; q is 2-4 and p is 0-1.
Particular compounds of the invention are;
2-methyl-3-nitrobenzoyl-Trp-piperazine-4-nitrophenyl;
2,4-dimethyl-benzoyl-Trp-piperazine-4-nitrophenyl;
5-bromo-2-furoyl-Trp-piperazine-4-nitrophenyl;
2-methoxybenzoyl-Trp-piperazine-4-nitrophenyl;
dimethoxynitrobenzoyl-Trp-piperazine-4-nitrophenyl;
2-methyl-3-furoyl-Trp-piperazine-4-nitrophenyl;
tiophene-3-carboxyl-Trp-piperazine-4-nitrophenyl;
2-(methylthio)-nicotinoyl-Trp-piperazine-4-nitrophenyl;
3-chlorothiphene-2-carboxyl-Trp-piperazine-4-nitrophenyl;
2,5-dimethoxybenzoyl-Trp-piperazine-4-nitrophenyl;
2-benzofuroyl-Trp-piperazine-4-nitrophenyl;
N-methylpyrrole-2-carboxyl-Trp-piperazine-4-nitrophenyl;
2-bromo-5-methoxybenzoyl-Trp-piperazine-4-nitrophenyl;
thiophene-2-carboxyl-Trp-piperazine-4-nitrophenyl;
5-chlorothiophene-2-carboxyl-Trp-piperazine-4-nitrophenyl;
2-methoxy-4-nitrobenzoyl-Trp-piperazine-4-nitrophenyl;
2-furylcarbonyl-Trp-piperazine-4-nitrophenyl;
6-methylpicoliny-Trp-piperazine-4-nitrophenyl;
3-methoxy-2-nitrobenzoyl-Trp-piperazine-4-nitrophenyl;
Dimethoxynitrobenzoyl-(D)Trp-piperazine-4-nitrophenyl;
4-oxo-4H-1-benzopyran-2-caboxyl-Trp-piperazine-4-nitrophenyl;
5-nitro-2-furoyl-Trp-piperazine-4-nitrophenyl;
1,2,3-thiadiazole-4-carboxyl-Trp-piperazine-4-nitrophenyl;
Z-Lys-(NMe)Phe-Trp-piperazine-4-nitrophenyl;
cyclohexylacetyl-(D)Lys-(D)NMe-Phe-(D)Trp-piperazine-4-nitrophenyl;
adamantaneacetyl-(D)Lys-(D)NMe-Phe-(D)Trp-piperazine-4-nitrophenyl;
cycloheptanoyl-(D)Lys-(D)NMe-Phe-(D)Trp-piperazine-4-nitrophenyl;
2-propylpentanoyl-(D)Lys-(D)NMe-Phe(D)Trp-piperazine-4-nitrophenyl;
Z-(D)NMe-Lys-(D)NMe-Phe-(D)Trp-piperazine-4-nitrophenyl;
Me2CHCH2(D,L)NME-Phe{CH2NH}(D)Trp-piperazine-4-nitrophenyl;
Z-LYS-(NMe)Phe-(D)Trp-piperazine-4-nitrophenyl;
cyclohexyl-(D)Lys-(D)(NMe)Phe-(D)Trp-piperazine-4-nitrophenyl;
T-butylacetyl-(D)Lys-(D)NMePhe-(D)Trp-piperazine-4-nitrophenyl;
cyclohexyl-(D)Lys-(D)NMe-Phe-(D)Phe(4-Cl)-piperazine-dichlorophenyl;and
cyclohexyl-(D)Lys-(D)NMePhe-(D)Tyr(Et)-piperazine-4-nitrophenyl;
or a pharmaceutically acceptable salt, prodrug or solvate thereof.
Further particular compounds of the invention are:
4,5-dimethoxy-2-nitrobenzoyl-Phe(4-Cl)-piperazine-4-nitrophenyl;
4,5-dimethoxy-2-nitrobenzoyl-NMe-Trp-piperazine-4-nitrophenyl;
4,5-dimethoxy-2-nitrobenzoyl-(D)(Nin-Me)Trp-piperazine-4-nitrophenyl;
Z-(D)(NMe)Dab-(NMe)(D)Phe-(D)Trp-piperazine-4-nitrophenyl;
Z-NMe(D)Lys-NMe(D)Phe-(D)Phe(4-Cl)-piperazine-4-nitrophenyl;
cyclohexyl-CO-(D)Lys-(D)NMe-Phe-Cha-piperazine-4-nitrophenyl;
cyclohexyl-(D)Lys-(D)(NMe)Phe-(D)Phe(4-Cl)-piperazine-4-nitrophenyl;
cyclohexyl-CH2-(D,L)NMe-Phe{CH2NH}(D)Trp-piperazine-4-nitrophenyl; and
cyclohexyl-(D)Lys-(D)(NMe)Phe-(D)hPhe-piperazine-4-nitrophenyl;
or a pharmaceutically acceptable salt, prodrug or solvate thereof.
A suitable pharmaceutically-acceptable salt of a compound of the formula (1) is, for example, an acid-addition salt of a compound of the Formula (1) which is sufficiently basic, for example an acid-addition salt with an inorganic or organic acid such as hydrochloric, hydrobromic, sulphuric, trifluoroacetic, citric or maleic acid; or, for example a salt of a compound of the Formula (1) which is sufficiently acidic, for example an alkali or alkaline earth metal salt such as a calcium or magnesium salt, or an ammonium salt, or a salt with an organic base such as methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris-(2-hydroxyethyl)amine.
Various forms of prodrugs are known in the art, for example in-vivo hydrolysable esters.
In vivo hydrolysable derivatives include, in particular, pharmaceutically acceptable derivatives that may be oxidised or reduced in the human body to produce the parent compound or esters that hydrolyse in the human body to produce the parent compound. Such esters can be identified by administering for example, intravenously to the test animal, the compound under test and subsequently examining the test animal""s body fluids. Suitable in vivo hydrolysable esters for hydroxy include acetyl and for carboxyl include, for example, alkyl esters, dialkylaminoalkoxy esters, esters of formula xe2x80x94C(O)xe2x80x94Oxe2x80x94CH2C(O)NRaxe2x80x3Rbxe2x80x3 where Raxe2x80x3 and Rbxe2x80x3 are, for example, selected from hydrogen and C1-4 alkyl, and C1-6alkoxy methyl esters for example methoxymethyl, C1-6alkanoyloxymethyl esters for example pivaloyloxymethyl, phthalidyl esters, C3-8 cycloalkoxycarbonyloxyC1-6alkyl esters for example 1-cylohexylcarbonyloxyethyl; 1,3-dioxolan-2-ylmethyl esters for example 5-methyl-1,3-dioxolan-2-ylmethyl; and C1-6-alkoxycarbonyloxyethyl esters for example 1-methoxycarbonyloxyethyl.
For examples of prodrug derivatives see:
a) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985) and Methods in Enzymology, Vol. 42, p. 309-396, edited by K. Widder, et al. (Academic Press, 1985);
b) A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen and H. Bundgaard, Chapter 5 xe2x80x9cDesign and Application of Prodrugsxe2x80x9d, by H. Bundgaard p. 113-191 (1991);
c) H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992);
d) H. Bundgaard, et al., Journal of Pharmaceutical Sciences, 77, 285 (1988); and
e) N. Kakeya, et al., Chem Pharm Bull, 32, 692 (1984).
It will also be understood that certain compounds of the present invention may exist in solvated, for example hydrated, as well as unsolvated forms. It is to be understood that the present invention encompasses all such solvated forms which possess the property of inhibiting MDM2 interactions.
In another aspect the present invention provides a process for preparing a compound of the Formula (1) or a pharmaceutically acceptable salt, prodrug or ester thereof which process comprises deprotecting a compound of the Formula (7): 
wherein n, L1 and R4 are as hereinabove defined and R1xe2x80x2 is R1 or protected R1, R2xe2x80x2 is R2 or protected R2, R3xe2x80x2 is R3 or protected R3 and R5xe2x80x2 is R5 or protected R5; wherein at least one protecting group is present; and thereafter if necessary:
i) forming a pharmaceutically-acceptable salt,
ii) forming a prodrug, and/or
iii) forming a solvate.
Protecting groups may in general be chosen from any of the groups described in the literature or known to the skilled chemist as appropriate for the protection of the group in question, and may be introduced by conventional methods.
Protecting groups may be removed by any convenient method as described in the literature or known to the skilled chemist as appropriate for the removal of the protecting group in question, such methods being chosen so as to effect removal of the protecting group with minimum disturbance of groups elsewhere in the molecule.
Specific examples of protecting groups are given below for the sake of convenience in which xe2x80x9clowerxe2x80x9d signifies that the group to which it is applied preferably has 1-4 carbon atoms. It will be understood that these examples are not exhaustive. Where specific examples of methods for the removal of protecting groups are given below these are similarly not exhaustive. The use of protecting groups and methods of deprotection not specifically mentioned is of course within the scope of the invention.
A carboxy protecting group may be the residue of an ester-forming aliphatic or araliphatic alcohol or of an ester-forming silanol (the said alcohol or silanol preferably containing 1-20 carbon atoms).
Examples of carboxy protecting groups include straight or branched chain C1-12alkyl groups (for example isopropyl, t-butyl); lower alkoxy lower alkyl groups (for example methoxymethyl, ethoxymethyl, isobutoxymethyl); lower aliphatic acyloxy alkyl groups, (for example acetoxymethyl, propionyloxymethyl, butyryloxymethyl, pivaloyloxymethyl); lower alkoxycarbonyloxy lower alkyl group (for example 1-methoxycarbonylethyl, 1-ethoxycarbonylethyl); phenyl lower alkyl groups (for example benzyl, p-methoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, benzhydryl and phthalidyl); tri(lower alkyl)silyl groups (for example trimethylsilyl and t-butyldimethylsilyl); tri(lower alkyl)silyl lower alkyl groups (for example trimethylsilylethyl); and C2-6alkenyl groups (for example allyl and vinylethyl).
Methods particularly appropriate for the removal of carboxy protecting groups include for example acid-, base-, metal- or enzymically-catalysed hydrolysis.
Example of hydroxy protecting groups include lower groups (for example t-butyl), lower alkenyl groups (for example allyl); lower alkanoyl groups (for example acetyl); lower alkoxycarbonyl groups (for example t-butoxycarbonyl); lower alkenyloxycarbonyl groups (for example allyloxycarbonyl); phenyl lower alkoxycarbonyl group (for example benzoyloxycarbonyl, p-methoxybenzyloxycarbonyl, o-nitrobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl); tri lower alkylsilyl (for example trimethylsilyl, t-butyldimethylsilyl) and phenyl lower alkyl (for example benzyl) groups.
Examples of amino protecting groups include formyl, aralkyl groups (for example benzyl and substituted benzyl, p-methoxybenzyl, nitrobenzyl and 2,4-dimethoxybenzyl, and triphenylmethyl); di-p-anisylmethyl and furylmethyl groups; lower alkoxycarbonyl (for example t-butoxycarbonyl); lower alkenyloxycarbonyl (for example allyloxycarbonyl); phenyl lower alkoxycarbonyl groups (for example benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, o-nitrobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl; trialkylsilyl (for example trimethyisilyl and t-butyldimethylsilyl); alkylidene (for example methylidene); benzylidene and substituted benzylidene groups.
Methods appropriate for removal of hydroxy and amino protecting groups include, for example, acid-, base-, metal- or enzymically-catalysed hydrolysis, for groups such as p-nitrobenzyloxycarbonyl, hydrogenation and for groups such as o-nitrobenzyloxycarbonyl, photolytically.
The reader is referred to Advanced Organic Chemistry, 4th Edition, by Jerry March, published by John Wiley and Sons 1992, for general guidance on reaction conditions and reagents. The reader is referred to Protective Groups in Organic Synthesis 2nd Edition, by Green et al, published by John Wiley and Sons for general guidance on protecting groups.
Compounds of the formula (1) and (7) can be formed by:
i) reacting a carboxylic acid of formula (8) or a reactive derivative thereof with a piperazine of formula (9) 
wherein L1, n, R4, R1xe2x80x2-R3xe2x80x2 and R5xe2x80x2 are as hereinabove defined.
A suitable reactive derivative of an acid of the Formula III is, for example, an acyl halide, for example an acyl chloride formed by the reaction of the acid and an inorganic acid chloride, for example thionyl chloride; a mixed anhydride, for example an anhydride formed by the reaction of the acid and a chloroformate such as isobutyl chloroformate; an active ester, for example an ester formed by the reaction of the acid with a phenol such as pentafluorophenol, with an ester such as pentafluorophenyl trifluoroacetate or with an alcohol such as N-hydroxybenzotriazole; an acyl azide, for example an azide formed by the reaction of the acid and an azide such as diphenylphosphoryl azide; an acyl cyanide, for example a cyanide formed by the reaction of an acid and a cyanide such as diethylphosphoryl cyanide; or the product of the reaction of the acid and a carbodiimide such as dicyclohexylcarbodiimide.
The reaction between compounds of the formulae (8) and (9) is carried out under conditions known for peptide bond formation. Typically the reaction is carried out in an organic solvent such as DMF or DMA. A small amount of THF and DMSO may be added as well. The reaction is usually carried out in the presence of an activating agent such as O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) and a base, particularly an amine base, for example diisopropylethylamine. In addition 1-hydroxy-7-azabenzotriazole (HOAT) may be added as catalyst. The reaction is normally carried out in a temperature range of 0 to 80xc2x0 C., preferably at around ambient temperature. 2-(1H-Benzotriazol-1yl)-1,1,3,3-tetramethyluroniunhexafluorophosphate (HBTU), may be used instead of HATU in which case 1-hydroxy-benzotriazole (HOBt) is generally used as catalyst.
The compound of the formula (8) is conveniently prepared by solid phase organic synthesis on a polymer resin. The resin is preferably one that can be removed with mild acid such as chlorotritylchloride resin. For example of other suitable polystyrene resins see the Nova Biochem Combinatorial Chemistry Catalogue February 1997.
The synthesis of a compound of the formula (8) is normally started with an amino acid which has a xe2x80x94(CH2)nR4 side chain. The amino acid functional groups not taking part in the reaction are protected by various functional groups. For example, the N-terminal and side chain amino groups may be protected by using 9-fluoroenylmethoxycarbonyl (Fmoc), t-butoxycarbonyl (Boc), biphenylisopropoxycarbonyl (Bpoc), 2-[3,5-dimethoxyphenyl]propyl-2-oxycarbonyl (Ddz), adamantyloxycarbonyl (Adoc), allyloxycarbonyl (Aloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl and various substituted benzyloxycarbonyl groups. These protected groups can be cleaved when required by the standard techniques (e.g. acid or base treatment, catalytic hydrogenolysis and Pd(0) treatment or zinc/acetic acid treatment). The choice of protecting group depends to a large extent on the type of resin used. When the resin is chlorotrityichoride the preferred amino protecting group is Fmoc.
The protected amino acid is then mixed with the resin so that its reacts with the resin through its carboxy group. When the resin is chlorotrityl resin, the reaction between amino acid and resin is conventionally carried out in an organic solvent such as DMF in the presence of a base such as DIPEA. The amino-protecting group is then removed so that the R5xe2x80x2 can be introduced if it is other than hydrogen. The protecting group cleavage reactions can be performed at temperatures in the range of 4xc2x0 C. to 40xc2x0 C. (preferably at or about ambient temperature) and over a period of time in the range of 10 minutes to 24 hours.
The introduction of the R5xe2x80x2 group when R5xe2x80x2 is of the formula R6C(O)xe2x80x94 is carried out using standard methods know for the formation of an amide bond, as shown for example in the presence of HBTU as described above.
When R6 is of the formula R7 the group R6COxe2x80x94 can gradually be built up with subsequent amide bond forming reactions. For example the group NH(L2)xe2x80x94CH(CH2Ph)xe2x80x94COxe2x80x94 may be introduced onto the amino acid attached to the resin by reacting a compound of the formula PN(L2)xe2x80x94CH(CH2Ph)COOH (wherein P is an amino-protecting group), with the latter compound under amide bond-forming conditions. The amino-protecting group can then be removed and the resulting compound reacted with a compound of the formula PN(L3)CH((CH2)nR8)COOH under similar conditions. Again the amino-protecting group can be removed and the resulting compound reacted with a compound of the formula R9(CH2)pA1xe2x80x94COOH. Alternatively the group R7COOH could be formed itself through subsequent amide-bond forming reactions and then introduced in one step onto the amino acid that is attached to the resin.
The resin is then removed, which in the case of chlorotrityl resin involves treating it with acetic acid and trifluoroethanol, to give a compound of the formula (8).
For further information on the formation of amide bonds see the procedures disclosed in xe2x80x9cSolid Phase Peptide Synthesis: A practical approachxe2x80x9d by Atherton and Sheppard (published by IRL press at Oxford University Press, 1989). xe2x80x9cSolid Phase Peptide Synthesisxe2x80x9d by Stewart and Young (published by the Pierce Chemical Company, Illinois, 1984), xe2x80x9cPrinciples of Peptide Synthesisxe2x80x9d (published by Springer-Verlag, Berlin, 1984), and a series of books xe2x80x9cAmino Acids, Peptides and Proteinsxe2x80x9d (volumes 1-25; volume 25 published in 1994) (published by the Royal Society of Chemistry, Cambridge, UK).
Similar reaction conditions can be used to synthesise a compound of the (8) without using a resin support.
When R5xe2x80x2 is of the formula R6CH2xe2x80x94, a compound of the formula (8) may be prepared by reacting together a compound of the formula R6C(O)H and the appropriate amine under conditions known for the formation of a Schiff""s base, followed by reduction of the Schiff""s base to the methylamino group. The reaction is typically carried out in an organic solvent such as DMF or an alcohol such as methanol or ethanol. Suitable reducing agents include sodium trisacetoxyborohydride, sodium cyanoborohydride and sodium borohydride.
A compound of the formula (9) is conveniently prepared by reacting piperazine, in which one nitrogen is protected, with a group of the formula: 
wherein L4 is a leaving group and R1xe2x80x2-R3xe2x80x2 are as hereinabove defined. Preferably the leaving group is chloro. The reaction is generally carried out in an inert organic solvent such as toluene or methylformamide, in the presence of an organic base such as triethylamine or DBU, in a temperature range of 80-200xc2x0 C., preferably at reflux.
Optional substituents in a compound of the formula (1) or (7) or intermediates in their preparation may be converted into other optional substituents. For example an hydroxy group could be alkylated to a methoxy group.
Various substituents may be introduced into compounds of the formulae (1) or (7) and intermediates in this preparation, when appropriate, using standard methods known in the art. For example, an acyl group or alkyl group may be introduced into an activated benzene ring using Friedel-Crafts reactions, a nitro group by nitration with concentrated nitric acid and concentrated sulphuric acid and bromination with bromine or tetra(n-butyl)ammonium tribromide.
It will be appreciated that, in certain steps in the reaction sequence to compounds of the formula (1), it will be necessary to protect certain functional groups in intermediates in order to prevent side reactions. Deprotection may be carried out at a convenient stage in the reaction sequence once protection is no longer required.,
In order to use a compound of the Formula (1), or a pharmaceutically-acceptable salt or in vivo cleavable ester thereof, for the therapeutic treatment (including prophylactic treatment) of mammals including humans, it is normally formulated in accordance with standard pharmaceutical practice as a pharmaceutical composition.
According to this aspect of the invention there is provided a pharmaceutical composition which comprises an amide derivative of the Formula (1), or a pharmaceutically-acceptable or in vivo cleavable ester thereof, as defined herein before in association with a pharmaceutically-acceptable diluent or carrier.
The compositions of the invention may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intramuscular dosing or as a suppository for rectal dosing).
The compositions of the invention may be obtained by conventional procedures using conventional pharmaceutical excipients, well known in the art. Thus, compositions intended for oral use may contain, for example, one or more colouring, sweetening, flavouring and/or preservative agents.
Suitable pharmaceutically-acceptable excipients for a tablet formulation include, for example, inert diluents such as lactose, sodium carbonate, calcium phosphate or calcium carbonate, granulating and disintegrating agents such as corn starch or algenic acid; binding agents such as starch; lubricating agents such as magnesium stearate, stearic acid or talc; preservative agents such as ethyl or propyl p-hydroxybenzoate, and anti-oxidants, such as ascorbic acid. Tablet formulations may be uncoated or coated either to modify their disintegration and the subsequent absorption of the active ingredient within the gastrointestinal tract, or to improve their stability and/or appearance, in either case, using conventional coating agents and procedures well known in the art.
Compositions for oral use may be in the form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin, or olive oil.
Aqueous suspensions generally contain the active ingredient in finely powdered form together with one or more suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as lecithin or condensation products of an alkylene oxide with fatty acids (for example polyoxethylene stearate), or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives (such as ethyl or propyl p-hydroxybenzoate, anti-oxidants (such as ascorbic acid), colouring agents, flavouring agents, and/or sweetening agents (such as sucrose, saccharine or aspartame).
Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil (such as arachis oil, olive oil, sesame oil or coconut oil) or in a mineral oil (such as liquid paraffin). The oily suspensions may also contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set out above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water generally contain the active ingredient together with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients such as sweetening, flavouring and colouring agents, may also be present.
The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, such as olive oil or arachis oil, or a mineral oil, such as for example liquid paraffin or a mixture of any of these. Suitable emulsifying agents may be, for example, naturally-occurring gums such as gum acacia or gun tragacanth, naturally-occurring phosphatides such as soya bean, lecithin, an esters or partial esters derived from fatty acids and hexitol anhydrides (for example sorbitan monooleate) and condensation products of the said partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening, flavouring and preservative agents.
Syrups and elixirs may be formulated with sweetening agents such as glycerol, propylene glycol, sorbitol, aspartame or sucrose, and may also contain a demulcent, preservative, flavouring and/or colouring agent.
The pharmaceutical compositions may also be in the form of a sterile injectable aqueous or oily suspension, which may be formulated according to known procedures using one or more of the appropriate dispersing or wetting agents and suspending agents, which have been mentioned above. A sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example a solution in 1,3-butanediol.
Suppository formulations may be prepared by mixing the active ingredient with a suitable non-irritating excipients which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Suitable excipients include, for example, cocoa butter and polyethylene glycols.
Topical formulations, such as creams, ointments, gels and aqueous or oily solutions or suspensions, may generally be obtained by formulating an active ingredient with a conventional, topically acceptable, vehicle or diluent using conventional procedures well known in the art.
Compositions for administration by insufflation may be in the form of a finely divided powder containing particles of average diameter of, for example, 30 xcexcm or much less, the powder itself comprising either active ingredient alone or diluted with one or more physiologically acceptable carriers such as lactose. The powder for insufflation is then conveniently retained in a capsule containing, for example, 1 to 50 mg of active ingredient for use with a turbo-inhaler device, such as is used for insufflation of the known agent sodium cromoglycate.
Compositions for administration by inhalation may be in the form of a conventional pressurised aerosol arranged to dispense the active ingredient either as an aerosol containing finely divided solid or liquid droplets. Conventional aerosol propellants such as volatile fluorinated hydrocarbons or hydrocarbons may be used and the aerosol device is conveniently arranged to dispense a metered quantity of active ingredient.
For further information on Formulation the reader is referred to Chapter 25.2 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.
The amount of active ingredient that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the host treated and the particular route of administration. For example, a formulation intended for oral administration to humans will generally contain, for example, from 0.5 mg to 2 g of active agent compounded with an appropriate and convenient amount of excipients which may vary from about 5 to about 98 percent by weight of the total composition. Dosage unit forms will generally contain about 1 mg to about 500 mg of an active ingredient. For further information on Routes of Administration and Dosage Regimes the reader is referred to Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.
The size of the dose for therapeutic or prophylactic purposes of a compound of the Formula (1) will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well known principles of medicine.
In using a compound of the Formula (1) for therapeutic or prophylactic purposes it will generally be administered so that a daily dose in the range, for example, 0.5 mg to 75 mg per kg body weight is received, given if required in divided doses. In general lower doses will be administered when a parenteral route is employed. Thus, for example, for intravenous administration, a dose in the range, for example, 0.5 mg to 30 mg per kg body weight will generally be used. Similarly, for administration by inhalation, a dose in the range, for example, 0.5 mg to 25 mg per kg body weight will be used. Oral administration is however preferred, particularly in tablet form. Typically, unit dosage forms will contain about 1 mg to 500 mg of a compound of this invention.
The compounds of this invention may be used in combination with other drugs and therapies used in the treatment of disease states which would benefit from the inhibition of the interaction of p53 and MDM2. For example, the compounds of the Formula (1) could be used in combination with drugs and therapies used in the treatment of cancers, including, but not limited to, breast cancer, sarcomas, osteosarcoma, testicular cancer or oesophageal cancer.
If formulated as a fixed dose such combination products employ the compounds of this invention within the dosage range described herein and the other pharmaceutically-active agent within its approved dosage range. Sequential use is contemplated when a combination formulation is inappropriate.
Although the compounds of the Formula (1) are primarily of value as therapeutic agents for use in warm-blooded animals (including man), they are also useful whenever it is required to inhibit the interaction of p53 and MDM2. Thus, they are useful as pharmacological standards for use in the development of new biological tests and in the search for new pharmacological agents.
In a further aspect of the invention there is provided a method of probing the biochemistry of MDM2 interactions using a compound of formula (1).
In a further aspect of the invention there is provided for use in the treatment of a warm-blooded animal, by therapy, a compound of formula (1), or a pharmaceutically acceptable salt, prodrug or solvate thereof as herein defined.
In a further aspect of the invention there is provided a method of treating cancers which comprises administering to a warm-blooded animal an effective amount of a compound of formula (4), 
wherein:
L1 is hydrogen or methyl;
R1 and R2 and R3 are each independently hydrogen, halo, nitro, cyano, carbamoyl, N-(C1-4alkyl )carbamoyl, NN-(diC1-4alkyl)carbamoyl or C1-4alkoxycarbonyl;
R4 is indole, N-(C1-4 alkyl) indole, C5-7carbocyclic ring or aryl, any of which can be optionally substituted on ring carbon atoms with up to three substituents each independently selected from halo, C1-4alkyl, or C1-4alkoxy;
R5 is hydrogen, C1-4alkyl, R6CH2xe2x80x94 or R6C(O)xe2x80x94;
R6 is aryl, heteroaryl, heterocyclyl, aminoC3-6alkyl, N-(C1-4alkyl)aminoC3-6alkyl, NN-(diC1-4alkyl)aminoC1-4alkyl, or R7; wherein the aryl, heteroaryl or heterocyclyl rings may be optionally substituted with up to three substituents independently selected from nitro, C1-4alkyl, C1-4alkoxy, halo, (C1-4alkyl)sulfanyl, C1-4alkoxycarbonyl, N-(C1-4alkyl)carbamoyl, NN-(diC1-4alkyl)carbamoyl, N-(C1-4alkyl)amino or NN-(diC1-4alkyl)amino;
wherein R7 is either a group of formula (5) of formula (6): 
wherein:
L2, L3 and L4 are each independently hydrogen or methyl;
R8 is amino, guanadino, imidazolo, any of which can be mono or di-N-substituted with C1-4alkyl;
A1 is oxygen or a direct bond;
R9 is a C5-8 membered mono-carbocyclic ring, a C6-10 membered bi-carbocyclic ring, C8-12 membered tri-carbocyclic ring, C5-7alkyl or aryl, any of which can be optionally mono, bi or tri substituted by C1-4 alkyl;
R10 is C1-6alkyl or a C3-8mono-carbocyclic ring;
R11 is hydrogen, halo, C1-4alkyl, or C1-4alkoxy;
R12 is hydrogen or methyl or ethyl or R12 together with L2 forms a C5-7 nitrogen-containing heterocyclic ring;
R13 is hydrogen or methyl ethyl or R13 together with L4 forms a C5-7 nitrogen-containing heterocyclic ring;
n is 0, 1 or 2;
p is 0, 1 or 2;
q is an integer from 1 to 6
r is 0, 1 or 2;
s is 0, 1 or 2;
provided that when R6 is aryl, heteroaryl, heterocyclyl, aminoC3-6alkyl, N-(C1-4alkyl)aminoC3-6alkyl or NN-(diC1-4alkyl)aminoC3-6alkyl then R5 is other than R6CH2xe2x80x94; or a pharmaceutically acceptable salt, prodrug or solvate thereof.
In a further aspect of the invention there is provided a method of treating cancers, mediated by wild type or altered MDM2, which comprises administering to a warm-blooded animal an effective amount of a compound of formula (4), or a pharmaceutically acceptable salt, prodrug or solvate thereof as herein defined.
In a further aspect of the invention there is provided the use of a compound of formula (4) or a pharmaceutically acceptable salt, prodrug or solvate thereof as herein defined, in the manufacture of a medicament for use in the treatment of cancer.
In a further aspect of the invention there is provided the use of a compound of formula (4) or a pharmaceutically acceptable salt, prodrug or solvate thereof as herein defined, in the manufacture of a medicament for use in the treatment of cancer, mediated by wild type or altered MDM2.
In a further aspect of the invention there is provided the use of a compound of formula (4) or a pharmaceutically acceptable salt, prodrug or solvate thereof as herein defined in the manufacture of a medicament for the treatment of cancer in a warm-blooded animal.
In a further aspect of the invention there is provided the use of a compound of formula (4) or a pharmaceutically acceptable salt, prodrug or solvate thereof as herein defined in the manufacture of a medicament for the treatment of cancers, mediated by wild-type or altered MDM2, in a warm-blooded animal.
In a further aspect of the invention there is provided a pharmaceutical composition comprising a compound of formula (4), or a pharmaceutically acceptable salt, prodrug or solvate thereof as herein defined, in admixture with a pharmaceutically-acceptable diluent or carrier for the treatment of cancer in a warm-blooded animal.
In a further aspect of the invention there is provided a pharmaceutical composition comprising a compound of formula (4), or a pharmaceutically acceptable salt, prodrug or solvate thereof, in admixture with a pharmaceutically-acceptable diluent or carrier for the treatment of cancer, mediated by wild-type or altered MDM2, in a warm-blooded animal.
The following biological assay provides a method of measuring the inhibition of the interaction of p53 with MDM2.
Measurement of the Inhibition of p53/MDM2 Interaction
The inhibition of the p53/MDM2 interaction by compounds was measured using a modified ELISA assay using a histidine tagged MDM2 [MDM2 (1-118) 6 his], a p53 GST fusion protein (GST-p53) and a nickel chelate-alkaline phosphatase (NTA-AP).
The human MDM2 N-terminal fragment encompassing amino acids 1-118 and immediately followed by a C-terminal 6-histidine tag sequence and then a stop codon was generated by PCR from a human placental cDNA library. The PCR product was initially cloned into pCR2.1 vector (Invitrogen) following the manufacturer""s protocol. DNA sequencing confirmed a MDM fragment sequence identical to that of published MDM2 (EMBL accession no. Z12020). The MDM2-6His fragment was then subcloned into pTB375 E. coli expression vector to produce the expression vector pTB375-MDM2-6His (1-118) clone no. 11. The pTB375 vector is a modified T7 (Studier) Expression vector. Expression grows were performed in E. coli strain BL21/DE3 (obtained from Novagen Inc.) and expression of the recombinant protein was induced by addition of IPTG (isopropyl-xcex2-D-1-thiogalactopyranoside). MDM2 (1-118) 6 his was purified from E. coli lysates using Ni-NTA beads (Qiagen).
The background vector for generation of pTB375 was pZEN0042 (pICI0042), described fully in UK patent application GB 2253852. Briefly, pZEN0042 contains the tetA/tetR inducible tetracycline resistance sequence from plasmid RP4 and the cer stability sequence from plasmid pKS492 in a pAT153 derived background. pTB375 was generated by the addition of an expression cassette consisting of the T7 gene 10 promoter, multiple cloning site and T7 gene 10 termination sequence. In addition, a terminator sequence designed to reduce transcriptional readthrough from the background vector was included upstream of the expression cassette.
A DNA fragment encoding aa1-50 of p53 followed by a stop codon was generated by PCR using vector pHp53B (ATCC clone no. 57255) as template. The PCR product was initially cloned into pCR2.1 vector (Invitrogen) following the manufacturer""s protocol. DNA sequencing confirmed a p53 sequence identical to that of published p53 (EMBL accession no. X04269).
The p53 fragment was then subcloned into pGEX-4T1 E coli expression vector (Pharmacia) to produce the expression vector pGEX-4T1-p53(1-50) clone no. 7. The sequence of the expression vector at the junction of the GST and p53 sequences was as follows:
GST seq . . . CTG GTT CCG CGT GGA TCC ATG . . . (SEQ ID NO: 1)
(ATG is aa1 of p53, GGA TCC is the conserved BamHI cloning site)
Expression grows were performed in E. coli strain DH5alpha and expression of the recombinant protein was induced by addition of IPTG. GST-p53(1-50) was purified using glutathione-sepharose beads (Pharmacia). The procedures used for expressing the GST fusion protein are therefore analogous procedures to those disclosed by J. Han et al., Journal of Biological Chemistry, 1996, 271, 2886-2891.
In the assay the following solutions were used:
Stock GST-p53 (6.1 mg/ml/xcx9c200 uM) diluted 2706 fold using H2O;
Stock MDM2-6His (1.1 mg/ml, xcx9c70 uM) diluted 933 fold using H2O;
Stock NTA-AP was made by reconstituting lyophilised Ni-NTA-AP (Qiagen 34510-200 xcexcg) in 500 ul distilled water and stored as frozen aliquots. It was frehly diluted 1:1000 using phosphate buffered saline (PBS) for use in the assay;
PNPP bufferxe2x80x94100 mM Tris HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl2;
2.5 mM Phosphatase substrate (Sigma 104 phosphatase substrate) was dissolved in PNPP buffer approximately 20 minutes prior to use; it was kept in the dark;
Double distilled water was used throughout the assay.
100 xcexcl of 75 nM GST-p53 was added per well to a 96 well plate (Nunc Maxisorp) and the plate left at 4xc2x0 C. overnight. Next day the fluid in the wells was removed and discarded and 100 xcexcl 0.2% bovine serum albumin (BSA) in PBS was added to each well and left at room temperature for 1 hour. The fluid was then removed and discarded and each well was washed twice with 0.1% Tween in PBS. After removing and discarding the second tween/PBS wash, 10 xcexcl of compound was added to each well, followed by 90 xcexcl 75 nM MDM2-6His. The plate was then incubated at room temperature for 1.5 hours. After this time the fluid from each well was removed and discarded and each well was washed three times with 280 xcexcl Tween/PBS. After removing and discarding the final wash, 100 xcexcl NTA-AP was added to each well and the plate incubated at room temperature for 1.5 hours. After this time the fluid from each well was removed and discarded and each well was washed three times with 280 xcexcl Tween/PBS. Next, 100 xcexcl phosphatase substrate in PNPP buffer was added and the plate left for 1-2 hours at 30xc2x0 C. The optical density of each well was measured at 405 nm.
Compounds were tested at a final concentration between 0 and 100 xcexcM. Stock solutions of compound were made at 30 xcexcM in DMSO, dilutions were made in H2O.
Although the pharmacological properties of the compounds of the Formula (1) vary with structural change as expected, in general compounds of the Formula (1) possess an IC50 in the above test in the range, for example, 0.03 to 200 xcexcM. Thus by way of example the compound of Example 9 herein has an IC50 of approximately 4 xcexcM.
The invention will now be illustrated by the following non-limiting Examples in which, unless otherwise stated:
(i) concentrations and evaporations were carried out by rotary evaporation in vacuo;
(ii) operations were carried out at room temperature, that is in the range 18-26xc2x0 C.;
(iii) yields, when given, are intended for the assistance of the reader only and are not necessarily the maximum attainable by diligent process development;
(iv) the following abbreviations are used:
BOC=tert-butoxycarbonyl; Cha=is an amino acid residue with a cyclohexylmethyl side chain; Dab=is an amino acid residue with a 2-aminoethyl side chain; DBU=1,8-diazabicyclo[5.4.0]undec-7-ene; DMF=N,N-dimethylformamide; HOBt=1-hydroxybenzotriazole; Met=methionine; Fmoc=9-fluorenylmethyloxycarbonyl; THF=tetrahydrofuran; DMSO=dimethylsulfoxide; HATU=O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HBTU=2-(1H-benzotriazol-1yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate; HOAT=1-hydroxy-7-azabenzotriazole; DIPEA=diisopropylethylamine; TFA=trifluoroacetic acid; HPLC=high pressure liquid chromatography; TCE=trichloroethane; RP-HPLC=reverse phase high pressure liquid chromatography (which unless otherwise stated was carried out on a Vydac C18 column 218TP54, 4.6xc3x97250 mm); and Z=benzyloxycarbonyl;
(v) flash chromatography and chromatography on silica were performed on Merck Kieselgel 60 (Art No. 9385) obtained from E Merck, Darmstadt, Germany; and
(vi) 1H NMR spectra were determined at 200 Mhz in CDCL3 or d6-dimethylsulphoxide (d6-DMSO) using tetramethylsilane (TMS) as an internal standard, and are expressed as chemical shift (delta) values in parts per million relative to TMS using conventional abbreviations for designation of major peaks: s, singlet; m, multiplet; t, triplet; br, broad, d, doublet.